ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.

Video Laryngoscopy Using King Vision™ aBlade™ and Direct Laryngoscopy in Paediatric Airway Management: A Randomized Controlled Study about Device Learning by Anaesthesia Residents.

Background: Airway management in children is challenging due to anatomical and physiological differences. This randomized trial investigates whether anaesthesia residents can intubate the paediatric trachea more quickly and with a higher success rate using the King Vision™ Paediatric aBlade™ video laryngoscope (KVL) compared to conventional direct laryngoscopy (DL). Methods: Eleven anaesthesia residents (mean age: 31 years, mean training status 47 months) were each asked to perform intubations with the KVL and DL in paediatric patients. The primary outcome was the first-attempt success rate. Secondary outcomes were the time to best view (TTBV), time to placement of the tracheal tube (TTP), time to ventilation (TTV), and participant-reported ease of use on a Likert scale. Results: 105 intubations with the KVL and 106 DL were performed by the residents. The success rate on the first attempt with the KVL was 81%, and the success rate on the first attempt within a given time limit of 30 s was 45%, which was lower than with DL (93% and 77% with time limit, p < 0.01). The median TTBV [IQR] on the first attempt with KVL was 7 [5−10] s, the median TTP was 28 [19−44] s, and the median TTV was 51 [39−66] s. DL-mediated intubation was significantly faster (TTP: 17 [13−23] s; p < 0.0001 and TTV: 34 [28−44] s; p < 0.001). Application of the KVL was rated as difficult or very difficult by 60% of the residents (DL: 5%). Conclusion: In contrast to promising data on the paediatric training manikin, residents took longer to intubate the airway in children with the KVL and were less successful compared to the DL. Therefore, the KVL should not be recommended for learning paediatric intubation by residents.

Inhibition of acyl-ACP thioesterase as site of action of the commercial herbicides cumyluron, oxaziclomefone, bromobutide, methyldymron and tebutam.

BACKGROUND: Understanding the mode and site of action of a herbicide is key for its efficient development, the evaluation of its toxicological risk, efficient weed control and resistance management. Recently, the mode of action (MoA) of the herbicide cinmethylin was identified in lipid biosynthesis with acyl-ACP thioesterase (FAT) as the site of action (SoA). Cinmethylin was registered for selective use in cereal crops for the control of grass weeds in 2020. RESULTS: Here, we present a high-resolution co-crystal structure of FAT in complex with cumyluron identified by a high throughput crystallization screen. We show binding to and inhibition of FAT by cumyluron. Furthermore, in an array of experiments consisting of FAT binding assays, FAT inhibition assays, physiological and metabolic profiling, we tested compounds that are structurally related to cumyluron and identified the commercial herbicides oxaziclomefone, methyldymron, tebutam and bromobutide, with so far unknown sites of action, as FAT inhibitors. Additionally, we show that the previously described FAT inhibitors cinmethylin and methiozolin bind to FAT in a nanomolar range, inhibit FAT enzymatic activity and lead to similar metabolic changes. CONCLUSION: Based on presented data, we corroborate cinmethylin and methiozolin as potent FAT inhibitors and identify FAT as the SoA of the herbicides cumyluron, oxaziclomefone, bromobutide, methyldymron and tebutam. © 2022 Society of Chemical Industry.

Functional PPO2 mutations: co-occurrence in one plant or the same ppo2 allele of herbicide-resistant Amaranthus palmeri in the US mid-south.

BACKGROUND: Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). RESULTS: The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS: Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.

Visualization of protein crystals by high-energy phase-contrast X-ray imaging.

For the extraction of the best possible X-ray diffraction data from macromolecular crystals, accurate positioning of the crystals with respect to the X-ray beam is crucial. In addition, information about the shape and internal defects of crystals allows the optimization of data-collection strategies. Here, it is demonstrated that the X-ray beam available on the macromolecular crystallography beamline P14 at the high-brilliance synchrotron-radiation source PETRA III at DESY, Hamburg, Germany can be used for high-energy phase-contrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix. Three-dimensional tomograms have been obtained at X-ray doses that are substantially smaller and on time scales that are substantially shorter than those used for diffraction-scanning approaches that display protein crystals at micrometre resolution. Adding a compound refractive lens as an objective to the imaging setup, two-dimensional imaging at sub-micrometre resolution has been achieved. All experiments were performed on a standard macromolecular crystallography beamline and are compatible with standard diffraction data-collection workflows and apparatus. Phase-contrast X-ray imaging of macromolecular crystals could find wide application at existing and upcoming low-emittance synchrotron-radiation sources.

Duration of exclusive breastfeeding is a positive predictor of iron status in 6- to 10-month-old infants in rural Kenya.

The prevalence of iron-deficiency anemia (IDA) is high in infants in Sub-Saharan Africa. Exclusive breastfeeding of infants to 6 months of age is recommended by the World Health Organization, but breast milk is low in iron. Some studies suggest exclusive breastfeeding, although beneficial for the infant, may increase risk for IDA in resource-limited settings. The objective of this study was to determine if duration of exclusive breastfeeding is associated with anemia and iron deficiency in rural Kenyan infants. This was a cross-sectional study of 6-10-month-old infants (n = 134) in southern coastal Kenya. Anthropometrics, hemoglobin (Hb), plasma ferritin (PF), soluble transferrin receptor (sTfR), and C-reactive protein were measured. Body iron stores were calculated from the sTfR/PF ratio. Socioeconomic factors, duration of exclusive breastfeeding, nature of complementary diet, and demographic characteristics were determined using a questionnaire. Mean ± SD age of the infants was 7.7 ± 0.8 months. Prevalence of anemia, ID, and IDA were 74.6%, 82.1%, and 64.9%, respectively. Months of exclusive breastfeeding correlated positively with Hb (r = 0.187; p < .05) and negatively with sTfR (r = -0.246; p < .05). sTfR concentrations were lower in infants exclusively breastfed at least 6 months compared with those exclusively breastfed for less than 6 months (7.6 (6.3, 9) vs. 8.9 (6.7, 13.4); p < .05). Controlling for gender, birth weight, and inflammation, months spent exclusively breastfeeding was a significant negative predictor of sTfR and a positive predictor of Hb (p < .05). The IDA prevalence in rural Kenyan infants is high, and greater duration of exclusive breastfeeding predicts better iron status and higher Hb in this age group.